Particulate acellular tissue matrix

ABSTRACT

A method of processing an acellular tissue matrix to give a particulate acellular tissue matrix includes: cutting sheets of dry acellular tissue matrix into strips; cryofracturing the dry acellular tissue matrix strips at cryogenic temperatures; separating the resulting particles by size at cryogenic temperatures; and freeze drying the fraction of particles desired size to remove any moisture that may have been absorbed to give a dry particulate acellular tissue matrix. Rehydration of the dry particulate acellular tissue matrix may take place just prior to use. The particulate acellular tissue may be applied to a recipient site, by way of injection, spraying, layering, packing, in-casing or combinations thereof. The particulate acelluar tissue may further include growth and stimulating agents selected from epidermal growth factor, fibroblast growth factor, nerve growth factor, keratinocyte growth factor, platelet derived growth factor, vasoactive intestinal peptide, stem cell factor, bone morphogetic proteins, chondrocyte growth factor and combinations thereof. Other pharmaceutically active compounds may be combined with the rehydrated particulate material including: analgesic drugs; hemostatic drugs; antibiotic drugs; local anesthetics and the like to enhance the acceptance of the implanted particulate material. The particulate material product may also be combined with stem cells selected from mesenchymal stem cells, epidermal stem cells, cartilage stem cells, hematopoietic stem cells and combinations thereof.

The present invention claims priority of U.S. Provisional PatentApplication No.: 60/089,865; Filed 19 Jun. 1998.

BACKGROUND OF THE INVENTION

Human, animal and synthetic materials are currently used in medicalprocedures to augment tissue or repair or correct tissue defects. To beoptimum, such materials should not migrate and should promote theregeneration of normal tissue, repopulating with the host's cells,revascularizing, and integrating with the patient's own tissue withouttriggering an inflammatory response that results in the degradation orresorption of the material. Additionally, the manner of delivery of suchmaterial, e.g. by surgical procedure or by injection, may significantlyaffect the clinical applications of the material, the ease of use by thephysician and the cost of the procedure.

Injectable collagen and other materials have been used clinically for awide variety of pathological and cosmetic applications in the fields ofreconstructive surgery, dermatology, oncology, otolaryngology andurology. Currently, the most widely used form of injectable collagen isderived from crosslinked bovine Type I collagen. In human clinicalapplications the effect of this xenogenic transplant is resorption bythe human host. Patients receiving these xenogenic grafts aresusceptible to an immune response to the animal collagen, requiringprescreening for existing antibodies. Examples of such materials may befound in U.S. Pat. Nos. 4,582,640; 5,104,957; 5,728,752; and 5,739,176.

Human collagen that may be injected is currently available and soldunder the tradenames Autologen® and Dermologen® and is manufactured byCollagenesis. This material it typically derived from autologouscollagen obtained during elective surgery or allogenic collagen fromcadavers. The starting material is dissociated by mechanical means andchemically treated to remove all noncollagenous proteins. The collagenis treated with additional chemicals to mask or crosslink the adverseeffects of these damaged and exposed collagen fibers. More informationwith regard to this technology may be found in U.S. Pat. Nos. 4,969,912and 5,332,802.

AlloDerm®, produced by LifeCell Corporation, is an acellular tissuematrix which is produced from normal human skin using processingtechniques established to remove the epidermis and cells within thedermis without significantly altering the normal biochemistry andmolecular architecture of the connective tissue matrix. The resultingproduct is in a freeze-dried form allowing extended shelf life and easeof shipping without degradation or loss of the normal tissue matrixcomponents. AlloDerm® is used clinically to repair or replace damaged orinadequate tissues. Reported applications for AlloDerm® include: fullthickness burn injury, replacement of lost gingiva due to periodontaldisease, reconstructive surgical applications involving the replacementof lost tissue or restoration of normal surface contours of skin damageddue to injury or aging neurosurgical application to replace lost duraand in urological applications such as bladder slings and pelvic floorreconstruction. AlloDerm® has been reported to integrate at the graftsite where it is rapidly repopulated with the normal milieu of hostcells. A reported benefit of AlloDerm® is that it maintains thestructure and biochemistry of the tissue matrix, promoting normal tissueregeneration. Studies have indicated that AlloDerm® retains decorin,hyaluronic acid, chondroitin sulfates, nidogen, growth factors and otherbiochemical proteins present in normal soft tissues. Additionally,AlloDerm® is reported to contain the basement membranes of vascularchannels and the orientation of elastin and collagen fibers of thestarting dermal tissue. For these reasons it is believed that thestructure and biochemistry of the AlloDerm® matrix promotes tissueregeneration. Reducing sheet AlloDerm® to a particulate suitable forinjection should extend the beneficial properties of AlloDerm® toseveral new applications.

Methods presently used to produce currently available injectablecollagen materials include mechanical disruption of the startingmaterial in its wet, hydrated state. However, when such processes arecarried out on intact autograft, allograft or xenograft tissue, damageto the matrix occurs such that following transplantation a foreign bodyresponse and rapid resorption of the tissue matrix occurs. Further,microscopic and histological analysis of material processed in such amanner exhibit mechanical disruption of the collagen fibers. Mechanicaldisruption of dried human or animal tissue at non-cryogenic temperaturesis believed to create a similar disruption of the collagen fibers,resulting in a foreign body response by the recipient and resorption ofthe material.

Thus there exists an unmet need for a method of making an intactparticulate acellular tissue matrix from acellular tissues.

SUMMARY OF THE INVENTION

The present invention is generally directed to a method of processing anacellular tissue matrix to give a particulate acellular tissue matrix. Ageneral embodiment of the method of the present invention includes thesteps of: cryofracturing the dry acellular tissue matrix strips atcryogenic temperatures; and separating the resulting particles by sizeat cryogenic temperatures. In a prefered embodiment, the method of thepresent invention includes: cutting sheets of dry acellular tissuematrix into strips; cryofracturing the dry acellular tissue matrixstrips at cryogenic temperatures; separating the resulting particles bysize at cryogenic temperatures; and freeze drying the fraction ofparticles desired size to remove any moisture that may have beenabsorbed to give a dry particulate acellular tissue matrix. Rehydrationof the dry particulate acellular tissue matrix may take place just priorto use.

It is generally preferred that the cryofracturing be carried out attemperatures below 0° C. and preferably the temperature should be belowabout −50° C. and more preferably should be below about −100° C.Commercially available refrigerants can be used to achieve suchtemperatures, which may include halocarbon refrigerants, liquid carbondioxide, liquid nitrogen, liquid argon, liquid helium and other similarsuch well known non-chemically reactive and thus inert and non-toxicrefrigerants. The separation of the resulting particles should also becarried out at cryogenic temperatures utilizing a series of metal meshscreens suitably sized for the particle range desired. In one preferredembodiment the screens are selected so as to isolate particles having asize of about 1 micron to about 900 microns and preferably screens areselected to isolate particles having a size from about 30 microns toabout 800 microns. The present invention also encompasses the product ofthe above described processes.

These and other features of the present invention are more fully setforth in the following description of illustrative embodiments of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The description is presented with reference to the accompanying drawingsin which:

FIG. 1 is an illustration of a bundle of collagen fibers which have beencryofractured in accordance with the present invention.

FIG. 2 is an illustration of a bundle of collagen fibers which have beenhomogenized at room temperature.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Acellular tissue matrix tissue is the result of a multistep process inwhich the tissue is collected from a donor and processed so as toisolate the natural tissue matrix. In its preferred form, the processincludes the steps of processing biological tissues including treatmentwith a stabilizing solution to reduce procurement damage, treatment witha processing solution to remove cells and other antigenic tissuecomponents, treatment with a cryoprotectant solution, freezing andstorage under specific conditions to avoid functionally significantdamaging ice crystal formation, drying under conditions to preventdamaging ice recrystallization, storage in the dry state at abovefreezing temperatures, rehydration under specific conditions and with arehydration solution to minimize surface tension damage and furtheraugment the selective preservation of the matrix, and reconstitutionwith viable cells that will not be rejected by the host.

The above summarized process for producing acellular dermal or othertissue matrix is more fully disclosed in U.S. Pat. No. 5,336,616, theentire contents of which is incorporated herein by reference.

One of skill in the art will appreciate however that other acellulartissue matrix tissues may be used in the present invention. For example,acellular tissue matrix tissue derived from xenogenic source may be usedin addition to the human derived tissue disclosed above, and othertissues such as blood vessels, heart valves, fascia and nerve connectivetissue may be used to create a particulate acellular matrix within thescope of the present invention. Thus in one preferred embodiment of thepresent invention, the method disclosed herein utilizes acellular tissuematrix tissue, herein also referred to as AlloDerm®, which iscommercially available from LifeCell Corporation, The Woodlands Texas.

The process of the present invention utilizes a chemical free andminimally disruptive technique which minimizes the damage to thecollagen fibers including sheared fiber ends that result from theconventional wet or dry processes previously disclosed. The resultingparticulate acellular tissue matrix can be suspended in a suitablecarrying agent and thereby is made suitable for delivery throughhypodermic needle injection or other modes of application including,spraying, layering, packing, in-casing or combinations of these methods.One of skill in the art should also appreciate that the particulateacellular matrix may be reconstituted into a sheet, or into a gelatinousform or other forms for use.

Generally the method of the present invention includes the steps of:cryofracturing the dry acellular tissue matrix strips at cryogenictemperatures; and separating the resulting particles by size atcryogenic temperatures. In a preferred embodiment, the method of thepresent invention includes: cryofracturing the dry acellular tissuematrix strips at cryogenic temperatures; separating the resultingparticles by size at cryogenic temperatures; and freeze drying thefraction of particles desired size to remove any moisture that may havebeen absorbed to give a dry particulate acellular tissue matrix. In amore preferred embodiment the method of the present invention includes:cutting sheets of dry acellular tissue matrix into strips;cryofracturing the dry acellular tissue matrix strips at cryogenictemperatures; separating the resulting particles by size at cryogenictemperatures; freeze drying the fraction of particles desired size toremove any moisture that may have been absorbed to give a dryparticulate acellular tissue matrix; and rehydrating the dry particulateacellular tissue matrix.

Preferably the dry acellular tissue matrix is cryogenically cooled to atemperature that permits the cryogenic fracturing, shattering or millingof the material. In one embodiment of the present invention a sterilizedhomogenizer cooled to liquid nitrogen temperatures is utilized. Theresulting cryogenically fractured material is then passed through aseries of particle size exclusion screens so as to isolate the desiredrange of particulate acellular tissue matrix material. Once isolated,the particulate acellular tissue matrix material may be freeze-dried soas to remove any moisture that may have been absorbed to the matrixduring the above described process.

Generally the particulate acellular tissue matrix is produced in such away as to minimize the amount of mechanical damage incurred whenreducing an intact tissue to a particulate form. As illustrated inFIG. 1. the cryofracturing of the collagen fibers in accordance with thepresent invention results in a “clean” break of the collagen fibers.This is in contrast with the frayed ends and substantially damagedcollagen fibers that result from the room temperature shredding ofcollagen tissue as is the practice in the prior art. The damage cause bythe mechanical shredding of the collagen fiber bundle, and in particularthe end of the collagen fibers is illustrated in FIG. 2. The aboveillustrations are based on electron microscopic observation of theprocessed materials and are representative of the ends of the collagenfiber bundles present in the collagen based tissues.

In developing the process of the present invention, several factors werefound to be important to the new and unexpected properties of theresulting particulate acellular tissue matrix. One such factor is thetemperature at which the homogenization or cryofracturing of the dryacellular matrix, such as AlloDerm®, takes place. As the term is usedherein, homogenization and cryofractuing are utilized interchangably andare intended to mean the process of creating particulate material fromthe sheet like starting material. It has been found that the temperatureat which the cryofractuing takes place should be sufficiently enoughbelow room temperature so that the collagen fibers are cryogenicallyfractured and not shredded or torn. Generally the temperature should bebelow 0° C. and preferably the temperature should be below about −50° C.and more preferably should be below about −100° C. Commerciallyavailable refrigerants can be used to achieve such temperatures, whichmay include halocarbon refrigerants, liquid carbon dioxide, liquidnitrogen, liquid argon, liquid helium and other similar such well knownnon-chemically reactive and thus inert and non-toxic refrigerants.

Another factor found to be important in achieving the present inventionis the particle size of the particulate acellular tissue matrixmaterial. In order to select the desired range of particles, acryogenically cooled homogenizing tower is utilized. The role of thehomogenization tower is to separate particles which are too small or toolarge in size from those within the desired particle size range. In oneembodiment, liquid nitrogen is utilized in combination with a firstmetal screen to reject those particles that are too large. A secondmetal screen is used in conjunction with liquid nitrogen to capturethose particles of the proper minimum size and to allow those particlesthat are too small to be removed. In one preferred embodiment the firstscreen is about 0.03 inch (0.0762 cm) metal screen and the second screenis about 0.0015 inch (0.00381 cm) metal screen. In another embodimentthe screens are selected so as to isolate particles having a size ofabout 1 microns to about 900 microns and preferably screens are selectedto isolate particles having a size from about 30 microns to about 800microns.

The resulting particulate acellular tissue matrix may be rehydrated bysuspension of the particles in any suitable aqueous solution, preferablynormal saline and local anesthetic. If desired the rehydratedparticulate acellular tissue matrix may be isolated by filtration orpelletizing the particle via centrifugation and decantation of thesupernatant. The particulate acellular tissue matrix may then beresuspended to an appropriate concentration in a suitablephysiologically compatible carrier, such as normal saline, normal salineand local anesthetic or if desired a pharmaceutical carrier. Either thephysiological carrier or the rehydrating saline solution may containantibiotics or other drugs, cells or cell extracts, anti-inflammatoryagents, proteoglycans, analgesics, hemostatic agents, growth factorssuch as epidermal growth factor, fibroblast growth factor, nerve growthfactor, keratinocyte growth factor, platelet derived growth factor,vasoactive intestinal peptide, stem cell factor, bone morphogenicproteins, chondrocyte growth factor and other similar such components aswell as other components that are desirable at the injection site.

Once rehydrated, the particulate acellular tissue matrix may also becombined with stem or progenitor cells prior to or duringtransplantation into the host. These stem cells may be native to thesite of transplantation, but due to their nature need not be. One ofordinary skill in the art should understand and appreciate that upondivision, stem cells replicate and also give rise to cells thatdifferentiate further into one or more specialized cells. Such stemcells may include mesenchymal stem cells, epidermal stem cells,cartilage stem cells, hematopoietic stem cells, and other similar cells.

Thus in one illustrative embodiment of the present invention theprocessing of acellular tissue matrix to create injectable sizeparticles includes: cutting sheets of dry acellular tissue matrix intostrips using a modified cutting or meshing devise; homogenization of thedry acellular tissue matrix strips at cryogenic temperatures; separationof the resulting particles by size at cryogenic temperatures; and freezedrying the fraction of particles desired to remove any moisture that mayhave been absorbed during homogenizing.

Another illustrative embodiment of the present invention includes theprocessing of acellular tissue derived from a human donor, e.g.AlloDerm®, to create injectable size particles includes: cutting sheetsof dry acellular tissue matrix into strips using a modified cutting ormeshing devise; equilibration of homogenizing equipment to liquidnitrogen temperatures; addition of dry acellular tissue matrix strips toa liquid nitrogen cooled homogenizer; cryofracturing of the dryacellular tissue matrix strips at liquid nitrogen temperatures;separation of the resulting particles by size at liquid nitrogentemperatures; and freeze drying the fraction of particles desired toremove any moisture that may have been absorbed during homogenizing.

Potential applications of the particulate acellular tissue matrixmaterials disclosed herein may include: dermatological applications suchas acne scar revision, replacement of dermis lost to disease oraccident; urological applications such as the relief of incontinence,and vesicoureteral reflux; otolaryngological applications includingvocal cord position adjustment; reconstructive surgical applications toreplace tissue lost to cancer surgery or other surgical procedures inwhich there is the removal of tissue; cosmetic surgery procedures suchas tissue replacement, reconstruction or augmentation procedures;correctional procedures for gastrointestinal reflux; and otherapplications which should be appreciated by one of skill in the art. Asan illustrative example, see U.S. Pat. No. 5,712,252, the contents ofwhich are hereby incorporated herein by reference and the referencescited therein.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the scope of theinvention.

Preparation Of Acellular Tissue Matrix

The following procedure was carried out in accordance with the teachingsof process for producing acellular dermal or other tissue matrix isfully disclosed in U.S. Pat. No. 5,336,616, the entire contents of whichis incorporated herein by reference, as well as being the subject ofco-pending U.S. Patent Applications including: 09/029,179, filed Aug.22, 1998; and the contents of which are hereby incorporated herein byreference. This material is commercially available from LifeCellCorporation, The Woodlands Texas.

Donor skin is harvested under aseptic conditions with a dermatome, andmaintained at 4° C. in RPMI 1640 tissue culture media containingpenicillin and streptomycin solution for no more than 7 days prior tofurther processing. Transportation to LifeCell's tissue processingcenter is via overnight delivery, on wet ice, in the same media. Onarrival at the processing center, the temperature of the tissuecontainer is verified to be at least 4° C., or the skin discarded.Following verification of container temperature, donor identificationand test screening data, the skin is transferred to a laminar-flow hoodfor further processing.

The donor skin is removed from the transportation container and placedwith its reticular side down on a piece of sizing support being a lowdensity polyethylene. An appropriately sized piece of gauze is added tothe epidermal side of the skin which is then cut into a rectangularpiece as large as possible, not to exceed a 4×4 inch square and nosmaller than 2×3 inches. The skin is then placed reticular side down, ina petri dish, to which 50 ml of De-epidermizing Solution consisting of1M NaCl is added. The petri dish is then transferred to an incubator andincubated at 37°±2° C. for 18 to 32 hours for human skin and 35 to 55hours for porcine skin.

After incubation, the petri dish containing the skin is transferred to alaminar flow hood for deepidermization. The gauze is first removed anddiscarded. The epidermis is then gently grasped with forceps and pulledaway from dermis as a sheet. The excess De-epidermizing Solution is thenaspirated. A slit approximately one centimeter long is then made in thelower left corner of the dermis to identify the upper and lowersurfaces.

The dermis is next rinsed in the same petri dish by the addition of 50ml Tissue Wash Solution, consisting of sterile Hanks' balanced saltsolution. The petri dish is then placed on a rotator at 40±5 RPM for 5minutes at room temperature (20°-26° C.). The petri dish is thenreturned to the laminar flow hood and the lid from the petri dish isremoved in order to aspirate the Tissue Wash Solution. This procedure isrepeated a further two times.

The dermis is then treated with 50 ml. of De-cellularizing solution andthe petri dish is placed on a rotator at 40±5 RPM for 1 hour at roomtemperature (20°-26° C.). The decellularizing solution for human skinconsists of 0.5% sodium dodecyl sulfate in Hanks' balanced salt solutionand for porcine skin contains 1 mM disodium ethylenediamine tetraaceticacid (EDTA). The De-cellularizing solution is removed by aspiration. Thedermis is then washed with 50 ml of Tissue Wash Solution. The petri dishis then placed on a rotator at 40±5 RPM for 5 minutes at roomtemperature (20°-26° C.). The Tissue Wash Solution is removed byaspiration. The washing procedure is repeated (2) times. After thedermis has been washed a total of 3 times 50 ml of Pre-freezing Solutionis added to the petri dish. The dish is then placed on a rotator at 40±5RPM for 30 minutes at room temperature (20°-26° C.). The prefreezingsolution for human skin consists of 7% dextran (70,000 MWT), 6% sucrose,6% raffinose and 1 mM disodium ethylenediamine tetraacetic acid inHanks' balanced salt solution. The prefreezing solution for porcine skinconsists of 7.5% dextran (70,000 MWT), 6% sucrose, 7.5%polyvinylpyrrolidone (MWT 40,000), 1.25% raffinose and 1 mM disodiumethylenediamine tetraacetic acid made up in Hanks' balanced saltsolution.

A new piece of gauze is then placed on the papillary side of the dermisand the dermis is turned over so that the reticular side faces up. Thebacking from the reticular side of the piece of dermis is discarded intoa biohazard waste container. An approximately 0.5 to 1.0 cm wide stripof backing and dermis is then cut from the original sample. This stripis then cut into two satellite pieces, each approximately 1.0 cm long.All necessary quality assurance is ultimately performed on thesesatellite samples, including microbiology and structural analysis.

The tissues are then transferred into individual TYVEK bags. The tissuesare positioned in the bag backing side up with the white vent side down.The TYVEK bag is then heat sealed.

The sealed Freeze-dry Bag is transferred to a freeze-dryer which has aminimum shelf temperature of −70° C. and a minimum condenser temperatureof −85° C. The tissue is then frozen on the freeze-dryer shelf byramping the shelf temperature at a rate of −2.5° C./minute to −35° C.,and held for at least 10 minutes.

The drying cycle is such that the final residual moisture content of thesample is less than 60% and optimally 2%. In this example, the frozendermis is dried by the following program:

1. The shelf temperature is ramped at a rate of −2.5° C./minute to −35°C., and held for 10 minutes, with vacuum set to 2000 mT (266 Pa).

2. The shelf temperature is then ramped at a rate of 1.5° C./minute to−23° C., and held for 36 hours with vacuum set to 2000 mT(266 Pa).

3. The temperature is then ramped at rate of 1.5° C./minute to a shelftemperature of −15° C., and held for 180 minutes with vacuum set to 2000mT(266 Pa).

4. The temperature is then ramped at a rate of 1.5° C./minute to a shelftemperature of −5° C. and held for 180 minutes with vacuum set to 2000mT(266 Pa).

5. The temperature is finally ramped at a rate of 1.5° C./minute to ashelf temperature of 20° C. and held for 180 minutes with the vacuum setto 0 mT(0 Pa).

Following drying, the Freeze-dry Bag containing the dried dermis isunloaded under an atmosphere of dry nitrogen gas, placed in a secondpredried impervious pouch and heat sealed under the same inertenvironment.

During the processing procedure and prior to sealing for freeze drying,a satellite sample is cut from the main sample and further processedunder identical conditions to the main sample. Prior to use of the mainsample in transplantation, all necessary quality assurance is performedon the satellite sample, including microbiology and structural analysis.

Following drying, the sample is stored at above freezing temperatures,optimally 4° C. in a light protected environment.

Preparation Of Particulate Acellular Tissue Matrix

The following procedure utilizes AlloDerm®, an acellular tissue matrixpackaged without a backing material the preparation of which isdescribed above. AlloDerm® is commercially available from LifeCellCorporation, The Woodlands Texas. After removal from the packaging, thedry, acellular tissue matrix is cut into strips using a Zimmer mesherfitted with a non-interrupting “continuous” cutting wheel. The resultinglong strips of acellular tissue matrix are cut into lengths of about 1to about 2 centimeters in length.

A homogenizer and sterilized homogenizer probe, such as a LabTeck Macrohomogenizer available from OMNI International, Warrenton, Va., isassembled and cooled to cryogenic temperatures using sterile liquidnitrogen which is poured into the homogenizer tower. Once thehomogenizer has reached cryogenic temperatures, acellular tissue matrixpreviously prepared into strips as noted above are added to thehomogenizing tower containing sterile liquid nitrogen. The homogenizeris then activated so as to cryogenically fracture the strips ofacellular tissue matrix. The time and duration of the cryogenicfractionation step will depend upon the homogenizer utilized, the sizeof the homogenizing chamber, the speed and time at which the homogenizeris operated and should be able to be determined by one of skill in theart by simple variation of the parameters to achieve the desiredresults.

The cryofractured particulate acellular tissue matrix material is sortedby particle size by washing the product of the homogenizer with liquidnitrogen through a series of metal screens, that have also been cooledto liquid nitrogen temperatures. We have found it especially useful toutilize a combination of screens within the homogenizing tower of thetype described above in which the particles are washed and sorted firstto exclude oversized particles and then to exclude undersized particles.

Once isolated, the particulate acellular tissue matrix is removed andplaced in a vial for freeze drying once the sterile liquid nitrogen hasevaporated. This last step is to ensure that any residual moisture thatmay have been absorbed during the above procedure is removed.

The final product is a white powder having a particle size of about 1micron to about 900 microns and preferably a particle size of about 30microns to about 750 microns. Preferably the particles are distributedabout a mean of about 150-300 microns. The material is readilyrehydrated by suspension in normal saline or other similar suitablerehydrating agent. The, rehydrated acellular tissue matrix may beresuspended in normal saline or any other suitable pharmaceuticallycompatible carrier.

Injection Of Particulate Acellular Tissue Matrix: A sample of the drycryofractured particulate acellular tissue matrix made in accordancewith the procedure disclosed hereinabove was rehydrated and resuspendedin phosphate buffer saline at a concentration of about 50 mg ofparticulate material per milliliter of phosphate buffered saline. Thesuspension was drawn into 1 cc tuberculin syringes. The samples weresent by overnight delivery at 4° C. to an independent laboratory forinjection into test animals. The samples were injected either into thedorsum of the back in the subcutaneous plane or subauricularly (on theback of the ears) of rats. The animals were monitored for 3 days, atwhich time samples of the skin surrounding and including the area ofinjection were excised for evaluation. Histological evaluation revealedparticles of human dermis just above the subcutaneous muscle layer inthe rat. Microscopic examination of the excised tissue revealed that theparticulate acellular tissue matrix had been repopulated with rat cellswith no evidence of severe acute inflammatory response by the hostanimal.

Comparison Of Wet Processed And Cryofractured Acellular Tissue Matrix:Samples of both the wet processed (room temperature) particulateacellular tissue matrix and the cryofractured (liquid nitrogentemperature) particulate acellular tissue matrix were prepared in thefollowing manner: Wet processed particulate acellular tissue matrix wasmade by first rehydrating a sample of AlloDerm® and cutting therehydrated acellular tissue matrix into strips and homogenizing thosestrips in phosphate buffered saline at room temperature. Thecryofractured particulate acellular tissue matrix was prepared inaccordance with the process of the present invention as describedhereinabove. Prior to use, the cryofractured particulate acellulartissue matrix was rehydrated in phosphate buffered saline and pelletedusing centrifugation.

Samples of each of the resulting particulate acellular tissue matrixmaterials were suspended in phosphate buffer saline at a concentrationof about 50 mg of particulate material per milliliter of phosphatebuffered saline. The suspension was drawn into 1 cc tuberculin syringes.The samples were sent by overnight delivery at 4° C. to an independentlaboratory for injection into test animals. Samples were injected eitherinto the dorsum of the back in the subcutaneous plane or subauricularlyinto rats. The animals were monitored for 3 weeks after which samples ofthe tissue surrounding and including the point of injection were excisedfor evaluation. Macroscopic and microscopic inspection of the samplesrevealed the following: Wet Processed Cryofractured Macroscopic 1/15*12/15* Microscopic 6/30* 24/30**# of samples exhibiting persistence of particulate AlloDerm ®/Total #of samples

Upon review of the above, one of skill in the art should understand andappreciate that the samples that were processed in accordance with thepresent invention had a rate of persistence approximately four timesthat of the wet processed material. From this, such a person shouldconclude that the wet processed particulate acellular tissue matrixcauses fundamental changes to the physiological properties of theacellular tissue matrix resulting in a more rapid degradation orresorption of the sample by the host animal.

Long Term Animal Study: Cryofractured particulate acelluar porcinetissue matrix prepared in accordance with the process of the presentinvention was injected subcutaneously and subdermally in a pig modelorganism. A concentration of 150 mg of particulate matrix per milliliterof saline was used in this study. Biopsies from the injection sites wereobtained at one, three and six months post-injection. These biopsiesrevealed evidence of persistence of the particulate matrix at 6 monthswith no evidence of acute inflammation. Further, the particulate matrixhad become repopulated with porcine fibroblasts and exhibited evidenceof revascularization of the particulate matrix.

In view of the above disclosure, one of ordinary skill in the art shouldappreciate that one illustrative embodiment of the present inventionincludes a method of processing an acellular tissue matrix to give aparticulate acellular tissue matrix, the method including:cryofracturing the dry acellular tissue matrix strips at cryogenictemperatures; and separating the resulting particles by size atcryogenic temperatures so as to produce the particulate acellular tissuematrix.

Another illustrative embodiment of the present invention includes amethod of processing an acellular tissue matrix to give a particulateacellular tissue matrix. The illustrative the method includes:cryofracturing the dry acellular tissue matrix strips at cryogenictemperatures; separating the resulting particles by size at cryogenictemperatures; and freeze drying the fraction of particles of desiredsize to remove any moisture that may have been absorbed so as to producethe particulate acellular tissue matrix.

Yet another illustrative embodiment of the present invention included amethod of processing an acellular tissue matrix to give a particulateacellular tissue matrix, the method comprising: cutting sheets of dryacellular tissue matrix into strips; cryofracturing the dry acellulartissue matrix strips at cryogenic temperatures; separating the resultingparticles by size at cryogenic temperatures; freeze drying the fractionof particles desired size to remove any moisture that may have beenabsorbed to give a dry particulate acellular tissue matrix; andrehydrating the dry particulate acellular tissue matrix.

In addition to the above methods, the present invention is also directedto the product of the processes described herein. In one illustrativeembodiment the product includes the product of the process disclosedherein along with growth and stimulating agents selected from epidermalgrowth factor, fibroblast growth factor, nerve growth factor,keratinocyte growth factor, platelet derived growth factor, vasoactiveintestinal peptide, stem cell factor, bone morphogenic proteins,chondrocyte growth factor and combinations thereof. Another embodimentof the present invention includes the product of the present inventioncombined with stem cells selected from mesenchymal stem cells, epidermalstem cells, cartilage stem cells, hematopoietic stem cells andcombinations thereof. The product of the present invention may alsofurther include analgesic drugs or hemostatic drugs or antibiotic drugsor combinations of these.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it should be apparent to those ofskill in the art that variations may be applied to the process describedherein without departing from the concept and scope of the invention.All such similar substitutes and modifications apparent to those skilledin the art are considered to be within scope and concept of theinvention.

1. A particulate acellular matrix comprising particles of an acellulartissue matrix, wherein the acellular tissue matrix is acellular anddehydrated and comprises a basement membrane to which, subsequent torehydration, viable endothelial cells or viable epithelial cellssecurely attach.
 2. The particulate acellular matrix of claim 21,further comprising one or more growth and stimulating agents selectedfrom the group consisting of: (a) epidermal growth factor, (b)fibroblast growth factor, (c) nerve growth factor, (d) keratinocytegrowth factor, (e) platelet derived growth factor, (f) vasoactiveintestinal peptide, (g) stem cell factor, (h) bone morphogenic proteins,and (i) chondrocyte growth factor.
 3. The particulate acellular matrixof claim 21, further comprising one or more stem cell populationsselected from the group consisting of (a) mesenchymal stem cells, (b)epidermal stem cells, (c) cartilage stem cells, and (d) hematopoieticstem cells.
 4. The particulate acellular matrix of claim 21, furthercomprising analgesic drugs.
 5. The particulate acellular matrix of claim21, further comprising hemostatic drugs.
 6. The particulate acellularmatrix of claim 21, further comprising antibiotic drugs.
 7. Theparticulate acellular matrix of claim 21, wherein the particles have asize of about 1 micron to about 900 microns.
 8. The particulateacellular matrix of claim 27, wherein the particles have a size of about30 microns to about 800 microns.
 9. The particulate acellular matrix ofclaim 21, wherein the acellular tissue matrix is made from dermis. 10.The particulate acellular matrix of claim 21, wherein the acellulartissue matrix is made from a tissue selected from the group consistingof blood vessel tissue, heart valve tissue, fascia, and nerve connectivetissue.
 11. The particulate acellular matrix of claim 21, wherein theacellular tissue matrix is made from a human collagen-based tissue. 12.The particulate acellular matrix of claim 21, wherein the acellulartissue matrix is made from a non-human animal collagen-based tissue. 13.The particulate acellular matrix of claim 32, wherein the non-humananimal is a pig.
 14. The particulate acellular matrix of claim 32,wherein the non-human animal is a dog.
 15. A method of treatment, themethod comprising applying the particulate acellular matrix of claim 21to a site in a recipient.
 16. The method of claim 35, wherein theapplying comprises one or more modalities selected from the groupconsisting of (a) injecting, (b) spraying, (c) layering, (d) packing,and (e) in-casing.
 17. The method of claim 35, wherein said recipienthas a defect selected from the group consisting of an acne scar, urinaryincontinence, vesicoureteral incontinence, lost tissue, andgastrointestinal reflux.
 18. The method of claim 37, wherein the losttissue is dermis.
 19. The method of claim 37, wherein the lost tissue istissue lost in surgery in which tissue is removed.
 20. A particulateacellular matrix comprising particles of an acellular tissue matrix,wherein the acellular tissue matrix is made by a process comprising: (a)procuring a collagen-based tissue; (b) incubating the collagen-basedtissue in a processing solution to produce processed tissue, wherein theprocessing solution extracts viable cells from the structural proteinand collagen matrix of the collagen-based tissue; (c) cryopreparing theprocessed tissue by incubation in a cryoprotective solution and freezingat cooling rates such that minimal functional damage occurs to thestructural protein and collagen matrix of the processed tissue toproduce a frozen, processed tissue; and (d) drying the frozen, processedtissue under temperature and pressure conditions that permit removal ofwater without substantial ice recrystallization or ultrastructuraldamage to produce a dried, processed tissue, wherein the residualmoisture content of the dried, processed tissue permits storage andrehydration of the dried, processed tissue.
 21. The particulateacellular matrix of claim 40, further comprising one or more growth andstimulating agents selected from the group consisting of: (a) epidermalgrowth factor, (b) fibroblast growth factor, (c) nerve growth factor,(d) keratinocyte growth factor, (e) platelet derived growth factor, (f)vasoactive intestinal peptide, (g) stem cell factor, (h) bonemorphogenic proteins, and (i) chondrocyte growth factor.
 22. Theparticulate acellular matrix of claim 40, further comprising one or morestem cell populations selected from the group consisting of (a)mesenchymal stem cells, (b) epidermal stem cells, (c) cartilage stemcells, and (d) hematopoietic stem cells.
 23. The particulate acellularmatrix of claim 40, further comprising analgesic drugs.
 24. Theparticulate acellular matrix of claim 40, further comprising hemostaticdrugs.
 25. The particulate acellular matrix of claim 40, furthercomprising antibiotic drugs.
 26. The particulate acellular matrix ofclaim 40, wherein the particles have a size of about 1 micron to about900 microns.
 27. The particulate acellular matrix of claim 46, whereinthe particles have a size of about 30 microns to about 800 microns. 28.The particulate acellular matrix of claim 40, wherein the collagen-basedtissue is dermis.
 29. The particulate acellular matrix of claim 40,wherein the collagen-based tissue is selected from the group consistingof blood vessel tissue, heart valve tissue, fascia, and nerve connectivetissue.
 30. The particulate acellular matrix of claim 40, wherein thecollagen-based tissue is a human collagen-based tissue.
 31. Theparticulate acellular matrix of claim 40, wherein the collagen-basedtissue is a non-human animal collagen-based tissue.
 32. The particulateacellular matrix of claim 51, wherein the non-human animal is a pig. 33.The particulate acellular matrix of claim 51, wherein the non-humananimal is a dog.
 34. A method of treatment, the method comprisingapplying the particulate acellular matrix of claim 40 to a site in arecipient.
 35. The method of claim 54, wherein the applying comprisesone or more modalities selected from the group consisting of (a)injecting, (b) spraying, (c) layering, (d) packing, and (e) in-casing.36. The method of claim 54, wherein said recipient has a defect selectedfrom the group consisting of an acne scar, urinary incontinence,vesicoureteral incontinence, lost tissue, and gastrointestinal reflux.37. The method of claim 56, wherein the lost tissue is dermis.
 38. Themethod of claim 56, wherein the lost tissue is tissue lost in surgery inwhich tissue is removed.